Helical Nucleocapsid Research Articles

Cryo-electron tomography of mouse hepatitis virus: Insights into the structure of the coronavirion

Coronaviruses are enveloped viruses containing the largest reported RNA genomes. As a result of their pleomorphic nature, our structural insight into the coronavirion is still rudimentary, and it is based mainly on 2D electron microscopy. Here we report the 3D virion structure of coronaviruses obtained by cryo-electron tomography. Our study focused primarily on the coronavirus prototype murine hepatitis virus (MHV). MHV particles have a distinctly spherical shape and a relatively homogenous size ( approximately 85 nm envelope diameter). The viral envelope exhibits an unusual thickness (7.8 +/- 0.7 nm), almost twice that of a typical biological membrane. Focal pairs revealed the existence of an extra internal layer, most likely formed by the C-terminal domains of the major envelope protein M. In the interior of the particles, coiled structures and tubular shapes are observed, consistent with a helical nucleocapsid model. Our reconstructions provide no evidence of a shelled core. Instead, the ribonucleoprotein seems to be extensively folded onto itself, assuming a compact structure that tends to closely follow the envelope at a distance of approximately 4 nm. Focal contact points and thread-like densities connecting the envelope and the ribonucleoprotein are revealed in the tomograms. Transmissible gastroenteritis coronavirion tomograms confirm all the general features and global architecture observed for MHV. We propose a general model for the structure of the coronavirion in which our own and published observations are combined.

Cell Type-Specific Cleavage of Nucleocapsid Protein by Effector Caspases during SARS Coronavirus Infection

The epidemic outbreak of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus (CoV), designated SARS-CoV. The RNA genome of SARS-CoV is complexed by the nucleocapsid protein (N) to form a helical nucleocapsid. Besides this primary function, N seems to be involved in apoptotic scenarios. We show that upon infection of Vero E6 cells with SARS-CoV, which elicits a pronounced cytopathic effect and a high viral titer, N is cleaved by caspases. In contrast, in SARS-CoV-infected Caco-2 cells, which show a moderate cytopathic effect and a low viral titer, this processing of N was not observed. To further verify these observations, we transiently expressed N in different cell lines. Caco-2 and N2a cells served as models for persistent SARS-CoV infection, whereas Vero E6 and A549 cells did as prototype cell lines lytically infected by SARS-CoV. The experiments revealed that N induces the intrinsic apoptotic pathway, resulting in processing of N at residues 400 and 403 by caspase-6 and/or caspase-3. Of note, caspase activation is highly cell type specific in SARS-CoV-infected as well as transiently transfected cells. In Caco-2 and N2a cells, almost no N-processing was detectable. In Vero E6 and A549 cells, a high proportion of N was cleaved by caspases. Moreover, we examined the subcellular localization of SARS-CoV N in these cell lines. In transfected Vero E6 and A549 cells, SARS-CoV N was localized both in the cytoplasm and nucleus, whereas in Caco-2 and N2a cells, nearly no nuclear localization was observed. In addition, our studies indicate that the nuclear localization of N is essential for its caspase-6-mediated cleavage. These data suggest a correlation among the replication cycle of SARS-CoV, subcellular localization of N, induction of apoptosis, and the subsequent activation of caspases leading to cleavage of N.

Nucleocapsid formation and RNA synthesis of Marburg virus is dependent on two coiled coil motifs in the nucleoprotein

The nucleoprotein (NP) of Marburg virus (MARV) is responsible for the encapsidation of viral genomic RNA and the formation of the helical nucleocapsid precursors that accumulate in intracellular inclusions in infected cells. To form the large helical MARV nucleocapsid, NP needs to interact with itself and the viral proteins VP30, VP35 and L, which are also part of the MARV nucleocapsid. In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor. Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system. Transfer of the coiled coil motif to a reporter protein was sufficient to mediate interaction of the constructed fusion protein with the N-terminus of NP. The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

Identification of mouse hepatitis coronavirus A59 nucleocapsid protein phosphorylation sites

The coronavirus nucleocapsid (N) is a multifunctional phosphoprotein that encapsidates the genomic RNA into a helical nucleocapsid within the mature virion. The protein also plays roles in viral RNA transcription and/or replication and possibly viral mRNA translation. Phosphorylation is one of the most common post-translation modifications that plays important regulatory roles in modulating protein functions. It has been speculated for sometime that phosphorylation could play an important role in regulation of coronavirus N protein functions. As a first step toward positioning to address this we have identified the amino acids that are phosphorylated on the mouse hepatitis coronavirus (MHV) A59 N protein. High performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was used to identify phosphorylated sites on the N protein from both infected cells and purified extracellular virions. A total of six phosphorylated sites (S162, S170, T177, S389, S424 and T428) were identified on the protein from infected cells. The same six sites were also phosphorylated on the extracellular mature virion N protein. This is the first identification of phosphorylated sites for a group II coronavirus N protein.

Acute peristome edema disease in juvenile and adult sea cucumbers Apostichopus japonicus (Selenka) reared in North China

Acute peristome edema disease (APED) is a new disease that broke out in cultured sea cucumber along the Shangdong and Liaoning province coasts in China, PR, and has caused a great deal of death in Apostichopus japonicus (Selenka) since 2004. Here we report virus-like particles found in intestine epithelium of sea cucumbers reared in North China. It is the first time that sea cucumbers are reported to be infected by virus. Histological examinations showed that the viral inclusion bodies existed in intestine epithelium cells. Electron microscopic examinations show that the virions were spherical, 80–100 nm in diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Detailed studies on the morphogenesis of these viruses found many characteristics previously described for coronaviruses. Virus particles always congregated, and formed a virus vesicle with an encircling membrane. The most obvious cellular pathologic feature is large granular areas of cytoplasm, relatively devoid of organelles. Tubular structures within virus-containing vesicles, nucleocapsid inclusions, and double-membrane vesicles are also found in the cytopathic cells. No rickettsia, chlamydia, bacteria, or other parasitic organisms were found.

Carboxyl Terminus of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Self-Association Analysis and Nucleic Acid Binding Characterization

Coronavirus nucleocapsid (N) protein envelops the genomic RNA to form long helical nucleocapsid during virion assembly. Since N protein oligomerization is usually a crucial step in this process, characterization of such an oligomerization will help in the understanding of the possible mechanisms for nucleocapsid formation. The N protein of severe acute respiratory syndrome coronavirus (SARS-CoV) was recently discovered to self-associate by its carboxyl terminus. In this study, to further address the detailed understanding of the association feature of this C-terminus, its oligomerization was systematically investigated by size exclusion chromatography and chemical cross-linking assays. Our results clearly indicated that the C-terminal domain of SARS-CoV N protein could form not only dimers but also trimers, tetramers, and hexamers. Further analyses against six deletion mutants showed that residues 343-402 were necessary and sufficient for this C-terminus oligomerization. Although this segment contains many charged residues, differences in ionic strength have no effects on its oligomerization, indicating the absence of electrostatic force in SARS-CoV N protein C-terminus self-association. Gel shift assay results revealed that the SARS-CoV N protein C-terminus is also able to associate with nucleic acids and residues 363-382 are the responsible interaction partner, demonstrating that this fragment might involve genomic RNA binding sites. The fact that nucleic acid binding could promote the SARS-CoV N protein C-terminus to form high-order oligomers implies that the oligomeric SARS-CoV N protein probably combines with the viral genomic RNA in triggering long nucleocapsid formation.

A Contemporary View of Coronavirus Transcription

Coronaviruses are a family of enveloped, plus-stranded RNA viruses with helical nucleocapsids and extraordinarily large genomes. The hallmark of coronavirus transcription is the production of multiple subgenomic mRNAs that contain sequences corresponding to both ends of the genome. (Transcription is defined as the process whereby subgenome-sized mRNAs are produced, and replication is the process whereby genome-sized RNA, which also functions as mRNA, is produced.) Thus, the generation of subgenomic mRNAs involves a process of discontinuous transcription. The aim of this minireview is to describe our current understanding of coronavirus replication and transcription. For more detailed information, the reader is directed to other recent reviews (25, 28, 44a, 49, 63, 74, 94).

Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270–370, is determined to 1.75Å resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin.

S1f2-4 Structural insights into packaging of the helical nucleocapsid from the structure of SARS coronavirus(S1-f2: "Functions and dynamics of protein systems in various aspect",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1f2-4 Structural insights into packaging of the helical nucleocapsid from the structure of SARS coronavirus(S1-f2: "Functions and dynamics of protein systems in various aspect",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

SR-Rich Motif Plays a Pivotal Role in Recombinant SARS Coronavirus Nucleocapsid Protein Multimerization

The nucleocapsid (N) protein of SARS coronavirus (SARS-CoV) is reported to function in encapsidating the viral genomic RNA into helical nucleocapsid, and its self-association is believed to be vital in coating the viral genomic RNA. Characterization of SARS-CoV N multimerization may thereby help us better understand the coronavirus assembly. In the current work, using the yeast two-hybrid technique, an unexpected interaction between residues 1-210 and 211-290 (central region) of the SARS-CoV N protein was detected, and SPR results further revealed that the SR-rich motif (amino acids 183-197) of SARS-CoV N protein is responsible for such an interaction. Chemical cross-linking and gel-filtration analyses indicated that the residues 283-422 of the SARS-CoV N protein have multimeric ability, although the full-length N protein is prone to exist predominantly as dimers. In addition, the multimeric ability of the C-terminal domain of SARS-CoV N protein could be weakened by the SR-rich motif interaction with the central region (amino acids 211-290). All of these data suggested that the SR-rich motif of the SARS-CoV N protein might play an import role in the transformation of the SARS-CoV N protein between the dimer and multimer during its binding to its central region for self-association or dissociation. This current paper will hopefully provide some new ideas in studying SARS-CoV N multimerization.

Structural disorder within the replicative complex of measles virus: functional implications

Measles virus belongs to the Paramyxoviridae family within the Mononegavirales order. Its non segmented, single stranded, negative sense RNA genome is encapsidated by the nucleoprotein (N) to form a helical nucleocapsid. This ribonucleoproteic complex is the substrate for both transcription and replication. The RNA-dependent RNApolymerase (L) binds to the nucleocapsid template via its co-factor, the phosphoprotein (P). In this review, we summarize the main experimental data pointing out the abundance of structural disorder within measles virus N and P.We also describe studies indicating that structural disorder is a widespread property in the replicative complex of Paramyxoviridae and, more generally, of Mononegavirales. The functional implications of structural disorder are also discussed. Finally, we propose a model where the flexibility of the disordered N and P domains allows the formation of a tripartite complex (N̊-P-L) during replication, followed by the delivery of N monomers to the nascent genomic RNA chain.

Sendai Virus RNA-dependent RNA Polymerase L Protein Catalyzes Cap Methylation of Virus-specific mRNA

The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5'-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756-2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1-1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis.

Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine

The nucleocapsid (N) protein of SARS-coronavirus (SARS-CoV) is the key protein for the formation of the helical nucleocapsid during virion assembly. This protein is believed to be more conserved than other proteins of the virus, such as spike and membrane glycoprotein. In this study, the N protein of SARS-CoV was expressed in Escherichia coli DH5α and identified with pooled sera from patients in the convalescence phase of SARS. A plasmid pCI-N, encoding the full-length N gene of SARS-CoV, was constructed. Expression of the N protein was observed in COS1 cells following transfection with pCI-N. The immune responses induced by intramuscular immunization with pCI-N were evaluated in a murine model. Serum anti-N immunoglobulins and splenocytes proliferative responses against N protein were observed in immunized BALB/c mice. The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein. More importantly, the immunized mice produced strong delayed-type hypersensitivity (DTH) and CD8 + CTL responses to N protein. The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses. The results indicate that the N protein may be of potential value in vaccine development for specific prophylaxis and treatment against SARS.

The 12 Å Structure of Trypsin-treated Measles Virus N–RNA

Recombinant measles virus nucleoprotein (N) was produced in insect cells where it bound to cellular RNA to form helical N–RNA structures. These structures were observed by electron microscopy but were too flexible for high-resolution image analysis. Removal of the C-terminal tail of N by trypsin treatment resulted in structures that were much more rigid and seemed more regular. Several methods of image analysis were employed in order to make a helical reconstruction of the digested N–RNA. During this analysis, it became clear that the apparently regular coils of digested N–RNA consisted of a series of closely related helical states. The iterative helical real space reconstruction method allowed the identification of two helical states for which a reconstruction could be calculated. The model with the highest resolution shows N monomers that consist of three domains and that are connected to their neighbours by two narrow connections, one close to the helical axis and another toward the middle of the monomers. There are no connections between N molecules in subsequent helical turns. After labelling the RNA in the structure with cis-platinum, the connection closest to the helical axis increased in density, suggesting the position of the RNA. The shapes of the monomers of the nucleoproteins of influenza virus, rabies virus (both determined before) and that of measles virus (determined here) are all similar, whereas the overall shapes of their respective N–RNAs (nucleocapsids) is very different. This is probably due to the position and number of the connections between the N subunits in the N–RNA, one for influenza virus allowing much flexibility, two for rabies virus at either end of the N molecules leading to ribbons and two for measles virus leading to the typical paramyxovirus helical nucleocapsid.

Ultrastructural characterization of SARS coronavirus.

Severe acute respiratory syndrome (SARS) was first described during a 2002–2003 global outbreak of severe pneumonia associated with human deaths and person-to-person disease transmission. The etiologic agent was initially identified as a coronavirus by thin-section electron microscopic examination of a virus isolate. Virions were spherical, 78 nm in mean diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Herein, we show that infection with the SARS-associated coronavirus resulted in distinct ultrastructural features: double-membrane vesicles, nucleocapsid inclusions, and large granular areas of cytoplasm. These three structures and the coronavirus particles were shown to be positive for viral proteins and RNA by using ultrastructural immunogold and in situ hybridization assays. In addition, ultrastructural examination of a bronchiolar lavage specimen from a SARS patient showed numerous coronavirus-infected cells with features similar to those in infected culture cells. Electron microscopic studies were critical in identifying the etiologic agent of the SARS outbreak and in guiding subsequent laboratory and epidemiologic investigations.

Closterovirus bipolar virion: evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5' region.

The long flexuous virions of the Closteroviridae have a unique bipolar architecture incorporating two coat proteins, with most of the helical nucleocapsid encapsidated by the major coat protein (CP) and a small portion of one end encapsidated by the minor coat protein (CPm). It is not known whether CPm encapsidates the genomic RNA and, if so, which end and what effects transition between the two coat proteins. Two other virus-encoded proteins, an HSP70 homolog (HSP70h) and an approximately 61-kDa protein, are required to augment virion assembly. In this work, we examine the in vivo encapsidation of Citrus tristeza virus by its CPm in the absence of CP. In the absence of other assembly-related proteins, CPm protected a family of 5' coterminal RNAs, apparently because of pausing at different locations along the genomic RNA. Most of the nucleocapsids formed by CPm were short, but a few were full-length and infectious. Mutations within the 5' nontranslated region demonstrated that the CPm origin of assembly overlaps the previously described conserved stem-and-loop structures that function as a cis-acting element required for RNA synthesis. Thus, in the absence of CP, the CPm encapsidation is initiated from the 5' end of the genomic RNA. Coexpression of HSP70h and the p61 protein with CPm in protoplasts restricted encapsidation to the 5' approximately 630 nucleotides, which is close to the normal boundary of the bipolar virion, whereas the presence of either HSP70h or the p61 protein alone did not limit encapsidation by CPm.

Characterization of N protein self-association in coronavirus ribonucleoprotein complexes

Mouse hepatitis virus (MHV) nucleocapsid (N) protein binds to the large, single-stranded, positive-sense viral genomic RNA to form a helical nucleocapsid structure in mature virions. In addition N protein binds the intracellular form of the genomic RNA, all of the MHV subgenomic mRNAs, and expressed non-MHV RNA transcripts to form ribonucleoprotein (RNP) complexes in infected cells. Among the intracellular viral RNP complexes, only the genomic RNP complex is packaged into virus particles. The present study demonstrated that N protein in the MHV virion nucleocapsid and in the intracellular genome-length RNP complex that bound to viral envelope M protein was tightly self-associated such that its association was retained even after extensive RNase A-treatment of the RNP complexes. The RNase A-resistant tight N protein association in the virion nucleocapsid was not mediated by an intermolecular disulfide bridge between N proteins. In contrast, N protein association in the majority of the intracellular RNP complexes was susceptible to RNase A-treatment. Because the RNP complexes that specifically interact with the M protein are selectively packaged into MHV particles, the present data suggested that there was a distinct difference between N protein association in viral genomic RNP complexes that undergo packaging into virus particles and the subgenomic RNP complexes that are not packaged into MHV particles.

Early events of SARS coronavirus infection in vero cells.

An isolate from a patient in the recent severe acute respiratory syndrome (SARS) outbreak in Singapore was used to infect Vero E6 cells. This study concentrated on the first 30 min of infection. It was discovered that the SARS coronavirus attached, entered, and uncoated the nucleocapsids, all within a 30‐min period. At 5 min after infection, several virus particles lined the Vero cell plasma membrane. Virus particles were at various stages of fusion at the cell surface, since entry was not a synchronised process. After entry (10 and 15 min), spherical core particles moved into the cytoplasm within large vacuoles. Quite surprising at such early stages of infection (20 min), a virus‐induced change in the infected cells was evident. The induction of myelin‐like membrane whorls was obvious within the same vacuoles as the core particles. The significance of this virus‐induced change is unknown at this stage. By 25–30 min postinfection (p.i.), the spherical core particles appeared to be disassociating and, in their place, doughnut‐shaped electron‐dense structures were observed. These could be the virus genomes together with the helical nucleocapsids. They were no longer in large vacuoles but packaged into smaller vacuoles in the cytoplasm, and occasionally in small groups. J. Med. Virol. 71:323–331, 2003. © 2003 Wiley‐Liss, Inc.

Fatal, virus-associated peripheral neuropathy and retinopathy in farmed Penaeus monodon in eastern Australia. I. Pathology.

Lesions are described in farmed Penaeus monodon affected with a previously unreported, fatal disease, 'peripheral neuropathy and retinopathy' (PNR). Outbreaks, associated with minor to heavy mortalities, occurred in 22 of 25 ponds on a farm in eastern Australia during the mid to late 1998/99 growout period. Moribund prawns, 5 to 26 g mean body weight, gathered at pond edges and were typically reddish in colour, lethargic, with mild to moderate epibiotic fouling and 1 or more partially amputated appendages. Histologically, there was mild to severe, focal to diffuse degeneration and necrosis of axons and their sheaths, together with associated glial cell apoptosis, in peripheral nerve fibres. Of the 3 appendage types examined systematically, these pathognomonic lesions were most common and severe in proximal antennal nerves and less common and severe in distal antennal nerves, antennular nerves and pereiopod nerves. Mild to severe, acute to chronic retinitis, associated with degeneration and necrosis of retinular cells and their axons, was also present in most clinically affected prawns. Transmission electron microscopy revealed moderate to large numbers of intracytoplasmic rod-shaped, helical nucleocapsids and enveloped virions, morphologically consistent with a yellow head-like virus, in putative glial cells in the antennal nerve, in the fasciculated zone of the eye and in putative sensory nerve cells of antennules. Immunohistochemical examination revealed lesions, but not histologically normal tissues, in peripheral nerves, eyes, lymphoid organ and vas deferens that consistently stained positively for a yellow head-related virus. The findings strongly suggest that a yellow head-related virus such as the Australian gill-associated virus (GAV) is causally associated with PNR. It is likely that PNR was not recognised during earlier investigations of mid-crop mortalities of farmed P. monodon in eastern Australia because appropriate peripheral nerves and eyes were not routinely examined histologically.

Substitution of Two Residues in the Measles Virus Nucleoprotein Results in an Impaired Self-Association

The nucleoprotein (N) of measles virus encapsidates viral genomic RNA to form a helical nucleocapsid. Its strong self-association is a major hurdle in determining its high-resolution structure using X-ray crystallography. We report the bacterial expression, purification, and characterization of a variant N that has lost its ability to form nucleocapsid-like structures after substitution of two residues by polar residues. Using immunoprecipitation, circular dichroism, and limited proteolysis studies, we show that this nucleoprotein retains a folding similar to wild-type N. Furthermore, the variant N binds the phosphoprotein, indicating that it retains biochemical relevance. We also present evidence indicating that the N-terminus of N lies at the surface of the nucleocapsid. Beyond the identification of one region of N involved in self-association, our results should facilitate structural studies of N using X-ray crystallography.